This present invention provides an advanced mutifunctional biochip (AMB) that combines integrated circuit elements, electro-optical excitation and detection systems, and molecular receptor probes in a self-contained integrated microdevice. Methods for the use of such devices in the detection and quantitation of biomolecules, and their application to diagnostic and therapeutic regimens are also provided.
Living systems possess exquisite recognition elements (e.g., antibody, enzyme, gene probes, etc.), often referred to as bioreceptors, which allow specific identification and detection of complex chemical and biological species. Biosensors exploit this powerful molecular recognition capability of bioreceptors. Due to the exquisite specificity of the DNA hybridization process, there is an increasing interest in the development of DNA bioreceptor-based analytical systems (Kumar et al., 1994; Eggers et al., 1994; Schena et al., 1995; Vo-Dinh et al., 1994; Stevenson et al., 1994; Isola et al., 1996; Alarie et al., 1992; Vo-Dinh et al., 1987a; 1987b; Vo-Dinh et al., 1991; Saiki et al., 1988; Graham et al., 1992; Steffan and Atlas, 1991; Vogelstein and Kinzler, 1992; Sambrook et al., 1989; Vo-Dinh et al., 1998a; 1998b; Isola et al., 1998; http://www.Affymetryx.com; http://www.Nanogen.com).
Most biosensors previously reported are based on fiber optic probes, glass and silica plates used as the probe substrates which are externally connected to a photo sensing system, which generally consists of a conventional detection device, such as a photomultiplier, or a charge-coupled device (CCD). In general, the sampling platform containing the probes is small (the sampling platform is often referred as to a xe2x80x9cDNA chipxe2x80x9d or xe2x80x9cgene chipxe2x80x9d), but the entire device containing excitation laser sources and detection systems (often a confocal microscope system) is relatively large, e.g. table-top size systems (e.g., the Affymetrix system). Nanogen has also developed a biochip system, but this device is mainly designed to move DNA with electric field manipulation, and has been used only for DNA samples. Until now, there is no truly integrated biochip system that comprises probes, samplers, detector as well as amplifier and logic circuitry on board and that is capable of multifunction diagnostics capability.
There is a strong interest in the development of non-radioactive bioreceptor probes using DNA, enzymes, and/or antibody probes for use in a wide variety of diagnostic and quantitative applications, such as identification of the causal agents of infectious disease, diagnosis and therapy of a variety of medical conditions, and detection of biomolecules in samples from industry, biotechnology, and the environment. The use of such techniques in the areas of agriculture, genetic engineering, agribiotechnology, and bioremediation is also contemplated to facilitate the detection and quantitation of a variety of macromolecules, including those of biological and microbiological origins.
One type of devices, often referred to as a xe2x80x9cbiochipxe2x80x9d combines semiconductor detection system with biotechnology-based probes, and has received increasing interest. The inventor has developed a variety of self-contained biochip devices and systems (e.g., U.S. patent application Ser. No. 08/979,672, filed Nov. 26, 1997; Intl. Pat. Appl. Ser. No. PCT/US98/25294, filed Nov. 25, 1998, and U.S. patent application Ser. No. 09/236,758, filed Jan. 25, 1999, the entire contents of each of which is incorporated herein by reference in its entirety). While such biochips (as well as other currently available biochip devices) have several detection channels, they are, however, designed to use only one specific type of bioreceptor at a time, and are therefore unsuitable for simultaneous multidetection of a plurality of species. While these earlier biochip systems may be used for detecting either an individual or a plurality of a particular biochemical species on a single chip at the same time (e.g., in detecting one or more polynucleotides or in detecting one or more polypeptides, they were not devised to detect multiple biochemical species at the same time on the same chip (i.e. the simultaneous detection of polypeptides and polynucleotides on a single chip).
Until now, most DNA biosensors previously reported are based on fiber optic probes, glass and silica plates used as the probe substrates which are externally connected to a photo sensing system, which generally consists of a conventional detection device, such as a photomultiplier, or a charge-coupled device (CCD). Although the probes on the sampling platform are small (often referred to as a xe2x80x98DNA chipxe2x80x99 or xe2x80x98gene chipxe2x80x99), the entire device containing excitation laser sources and detection systems (often a confocal microscope system) is relatively large, e.g., tabletop size systems. Although these systems have demonstrated their usefulness in genomics research and analysis, they are laboratory-oriented and involve relatively expensive equipment.
There is a critical demand for a rapid, simple, cost-effective technique for screening samples, such as blood or other clinical samples, for the presence of biomolecules (including polynucleotides, polypeptides, etc.) to assist in the diagnosis and treatment of medical diseases, including those caused by infectious pathogens, and the like, as well as provide efficient means for quantitating such molecules in pathology and forensics samples. The development of inexpensive screening analyses that would permit simultaneous analyses of multiple biological molecules would allow rapid detection and improved treatments of many illnesses, facilitate improvements in quality control and manufacturing, as well as provide rapid, affordable devices for detection of biomolecules in the areas of environmental contamination and remediation processes.
The development of rapid and effective screening tests for simultaneous assay of two or more different types of molecules (e.g., detecting antibodies and polynucleotides in a sample on a single biochip) would also reduce the cost of diagnostic testing services, biochemical analyses and assay systems, as well as overall costs in the health care industry. For example, a critical factor in medical diagnostics is rapid, selective, and sensitive detection of biochemical substances (protein, metabolites, nucleic acids), biological species or living systems (bacteria, virus or related components) at ultra-trace levels in biological samples (e.g., tissues, blood and other bodily fluids). To achieve the required level of sensitivity and specificity in detection, it is often necessary to use a biosensor that is capable to identify and differentiate a large number of biochemical constituents in complex samples. The development of a cost-effective biochip alternative to simultaneous quantitation of pluralities of differing biological molecules would be a revolutionary advance in the fields of analytical chemistry and medicine.
To that end, there is currently a strong need for a truly advanced multifunctional biochip system that comprises the necessary probes, samplers, detectors, amplifier and logic circuitry on a single biochip useful in the detection of pluralities of different biomolecular targets. Such a system would be useful in many environments, including inter alia, diagnostic laboratories, environmental sites, remediation or hazardous materials clean-up sites, physicians"" offices, health care clinics, hospitals, and even mobile analytical chemistry devices.
Likewise, there is also a need to extend the application of biochip-based sensors to the detection and quantitation of macromolecules other than polypeptides and DNAs. The development of sensors useful in the detection of molecules such as RNAs, peptide-nucleic acids (PNAs), ribozymes, antibodies, enzymes, and peptide fragments, would represent a significant advance in the art and provide new methods and devices for the detection of molecules of biological importance. Furthermore, because of recent advances in the molecular sciences, the ability to detect and quantitate biomimetics, and new classes of biologically-active molecules such as DNA adaptamers, aptamers, cyclodextrins, dendrimers, molecular imprints, and the like would benefit from the creation of biochip-based apparatus capable of detecting and quantitating these, and other biologically- and medically-relevant macromolecules.
There is also a distinct need for development of advanced multifunctional devices that permit the rapid, large-scale and cost effective analysis of pluralities of heterogeneous macromolecules, and permit the development of methods for detecting and quantitating multiple molecular species in mixed biological samples.
This present invention overcomes these and other limitations in the prior art by providing for the first time, an advanced multifunctional biochip (AMB) that is capable of simultaneous detection of two or more different biological macromolecules (or xe2x80x9ctargetsxe2x80x9d). These macromolecules may comprise a plurality of polynucleotides (including DNAs, PNAs and/or RNAs), a plurality of polypeptides, peptides, and/or proteins; a plurality of enzymes, antibodies, and/or receptors or antigens); a plurality of pathogens, organisms, microorganisms, and/or viruses, etc.); or a plurality of cells, cell types, tissues, organelles, organs, fluids, and/or other intracellular or extracellular components of a living cell. Alternatively, the biological macromolecules may be a combination of any of these or other biological compounds that may be detected using an AMB that comprises a plurality of receptor probes, orbiomimetic probes on a single device. Such design permits the simultaneous or sequential detection of a variety of targets using a single biochip device, and as such provides methods of detecting and quantitating a number of diverse biochemical compounds using a single device.
Operating Principle of the AMB Device
Biochip System
Biosensors and biochips involve two essential functions that integrate xe2x80x9cbiological recognitionxe2x80x9d and xe2x80x9csensing.xe2x80x9d The basic principle of an optical biosensor is to detect this molecular recognition and to transform it into an optical signal using a transducer. The biochip is a biosensor that involves the combination of integrated circuit elements, electro-optics excitation/detection system, and bioreceptor probes into a self-contained and integrated microdevice. An illustrative medical biochip device of the present invention may include: 1) an excitation light source, 2) a bioprobe, 3) a sampling platform, 4) one or more sensing elements, and 5) a signal amplification and treatment system. FIG. 1A, FIG. 1B, and FIG. 1C show schematic diagrams of the AMB device having different types of probes (polypeptides, antibody, nucleic acids, enzymes, tissues, organelles, and other receptor probes). Different possible embodiments of the AMB are schematically shown in FIG. 1A and FIG. 1B.
Biological Probes
The biochip has a unique multifunctional diagnostic capability due to the different types of bioprobes: polynucleotides such as PNA, RNA or DNA; polypeptides, such as proteins, peptides, antibodies, enzymes, and receptors; as well as tissues, organelles, and other receptor probes.
DNA Probes
Recently there has been an increasing interest in biosensor technology. Biosensors combine two important concepts that integrate xe2x80x9cbiological recognitionxe2x80x9d and xe2x80x9csensing.xe2x80x9d The basic principle of a biosensor is to detect this molecular recognition and to transform it into another type of signal using a transducer. The selected transducer may produce either an optical signal (i.e. optical biosensors) or an electrochemical signal (i.e. electrochemical biosensors). The bioreceptor may consist of an enzyme, an antibody, a gene fragment, a chemoreceptor, a tissue, an organelle or a microorganism.
The operation of gene probes is based on the hybridization process. Hybridization involves the joining of a single strand of nucleic acid with a complementary probe sequence (FIG. 2). Hybridization of a nucleic acid probe to DNA biotargets (e.g., gene sequences, bacteria, viral DNA) offers a very high degree of accuracy for identifying DNA sequences complementary to that of the probe. Nucleic acids strands tend to be paired to their complements in the corresponding double-stranded structure. Therefore, a single-stranded DNA molecule will seek out its complement in a complex mixture of DNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (i.e. gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridization or re-association of two complementary DNA strands include temperature, contact time, salt concentration, the degree of mismatch between the base pairs, as well as the length and concentration of the target and probe sequence(s).
Probes Based on cDNAs
The AMB device can also be used to monitor gene expression using cDNA probes. Genes, which are contained within in the DNA of a cell""s nucleus, contain codes that essentially are recipes for tens of thousands of proteins. The code-containing regions of the gene (exons), however, are often separated by much non-coding DNA (introns). A cDNA molecule is a laboratory-made version of a gene that contains only its information-rich regions; these molecules provide a way for genome researchers to fast-forward through the genome to biologically important areas.
cDNA molecules are made using molecules of RNA (similar to DNA) obtained from living cells. In the cell, expression of the information from DNA into a protein first requires transcription of DNA into nuclear RNA molecules. These nuclear RNAs have non-coding regions that are processed out in the course of forming cytoplasmic RNAs (messenger RNAs). Because mRNAs are too fragile to withstand laboratory manipulations, scientists make sturdy double-stranded copies called complementary (or copy) DNA, or cDNAs.
All DNA clones derived from a particular tissue constitute a library of clones representing the genes that were expressed when the source tissue was harvested. The analysis of libraries from many different tissues, obtained under a variety of physiological conditions, will be necessary to decipher the organ-specific patterns of gene expression.
Antibody Probes
Antibodies are the product of immune system cells (B cells) when those cells are exposed to antigenic substances or molecules. The antibodies produced following antigen exposure have recognition/binding sites for specific molecular structures (or substructures) of the antigen. Just as specific configurations of a unique key enable it to enter a specific lock, so in an analogous manner, an antigen-specific antibody xe2x80x9cfitsxe2x80x9d its unique antigen. Thus, an antigen-specific antibody interacts with its unique antigen in a highly specific manner, so that the total three-dimensional biochemical conformation of antigen and antibody are complementary. This molecular recognition feature of antibodies is the key to their usefulness in immnunosensors; molecular structural recognition allows one to develop antibodies that can bind specifically to chemicals, biomolecules, microorganism components, etc. Such antibodies may then be used as specific xe2x80x9cprobesxe2x80x9d to identify an analyte of interest that is often present in extremely small amounts, among a myriad of other chemical substances. Another property of antibodies of great importance to their role in immunosensors is the strength or avidity/affinity of the antigen-antibody interaction. Since the antigen-antibody surfaces lie in close proximity to one another, a variety of molecular interactions may take place and the overall strength of such interactions can be considerable, with correspondingly highly favorable association and equilibrium constants.
General immunoassay measurement strategies can be divided into three categories: direct, competitive (Tromberg et al., 1987), and sandwich assays (Vo-Dinh et al., 1987a; 1987b). FIG. 3 shows a schematic of the three strategies. Direct assays are performed by simply incubating the naturally fluorescent antigens with excess amounts of antigens. The sensitivity is directly proportional to the amount of antibody present and the response is directly proportional to the amount of antigen present. In competitive assays, fluorescent-labeled antigen is used to compete with unlabeled (non-fluorescent) antigen for a limited number of antibody binding sites. Antibody-bound antigen (labeled and unlabeled) is separated from free antigen and the fluorescence of the antibody-bound labeled antigen is measured. The signal intensity of the bound phase is inversely proportional to the concentration of the unlabeled antigen. The higher the fluorescent signal, the lower the unlabeled antigen concentration. Sandwich assays are performed by incubating the antigens with a primary (first) antibody that is present in excess concentrations. The antibody-antigen complex is then incubated with a second fluorescently labeled antibody that binds to the first antibody. Unbound labeled antibody is rinsed away and the bound labeled antibody is measured. Sensitivity is related to the amount of primary antibodies present with the response proportional to the antigen concentration.
Multichannel Sampling Platform
Immobilization of bioprobes on a multiarray (e.g., those of up to about 100 or about 200, or about 400, or even about 1000 or so channels) sampling platform can be performed onto a transducer detection surface to ensure optimal contact and maximum detection. When immobilized onto a substrate, the bioprobes are stabilized and, therefore, can be reused repetitively. In one illustrative embodiment, the hybridization is performed on an immobilized target or a probe molecule attached on a solid surface such as a nitrocellulose, a nylon membrane or a glass plate using one of the well-known methods for binding a nucleic acid molecule to a particular substrate or support. The method most commonly used for binding bioprobes to glass involves silanization of the glass surface followed by activation with carbodiimide or glutaraidehyde. Immobilization of the bioreceptor probes onto a substrate or membrane and subsequently attaching the membrane to the transducer detection surface is another approach that can be used.
Integrated Electro-optic Microchip System
The instrumental system developed for this work involved integrated electro-optic sensing photodetectors for the biosensor microchips. The inventor has developed the design of the electro-optic systems for the microchip detection elements, the fabrication of which is possible partly through the capability of fabricating multiple optical sensing elements and microelectronics on a single integrated circuit (IC). An example of such integration is a two-dimensional array of optical detector amplifiers integrated on a single IC chip.
The detailed design of an illustrative electro-optic microchip has been described in (U.S. patent application Ser. No. 08/979,672, incorporated herein by reference in its entirety). An important element in the development of the biochip involves the design and development of an IC electro-optic system for the microchip detection elements using the CMOS technology. With this technology, highly integrated biosensors are made possible partly through the capability of fabricating multiple optical sensing elements and microelectronics on a single IC. A two-dimensional array of optical detector-amplifiers was integrated on a single IC chip. Such an integrated microchip system is not currently available commercially.
The inventor and others have developed two types of biochips, one using phototransistors (Vo-Dinh et al., 1998a; 1998b) and the other using photodiode systems (Vo-Dinh, 1998a; 1998b). Exemplary biochip IC systems based on photodiode circuitry typically comprise 16 channels (e.g., in a 4xc3x974 array), or larger (e.g., a 100-channel system that comprises a 10xc3x9710 array). The biochips include a large-area, n-well integrated amplifier-photodiode array that has been designed as a single, custom integrated circuit (IC), fabricated for the biochip. This IC device is coupled to the multiarray sampling platform and is designed for monitoring very low light levels. The individual photodiodes have 900-xcexcm square size and are arrayed on a 1-mm spacing grid. The photodiodes and the accompanying electronic circuitry were fabricated using a standard 1.2-micron n-well CMOS process. The use of this type of standard process allows the production of photodiodes and phototransistors as well as other numerous types of analog and digital circuitry in a single IC chip. This feature is the main advantage of the CMOS technology in comparison to other detector technologies such as charge-coupled devices or charge-injection devices. The photodiodes themselves are produced using the n-well structure that is generally used to make resistors or as the body material for transistors. Since the anode of the diode is the p-type substrate material, which is common to every circuit on the IC chip, only the cathode is available for monitoring the photocurrent and the photodiode is constrained to operate with a reverse bias.
The inventor has designed an analog multiplexer that allows any of the elements in the array to be alternately connected to a single amplifier. Optionally each photodiode could be supplied with its own amplifier. The multiplexer for a 4xc3x974-array device may, therefore, be made from 16 cells. Each cell has two CMOS switches that are controlled by the output of the address decoder cell. Each cell has a unique 4-bit address. One switch is open only when it is being addressed while the other switches are closed. This process connects the addressed diode to one amplifier while all the others are connected in parallel to the other amplifier. This arrangement allows connecting a 4xc3x974 (or 10xc3x9710) array of light sources (different fluorescent probes, for example) to the photodiode array and reading out the signal levels sequentially. With some modification, a parallel reading system may also be utilized. Using a single photodiode detector would require mechanical motion to scan the source array. The additional switches and amplifier serve to correctly bias and capture the charge generated by the other photodiodes. The additional amplifier and switches allow the IC to be used as a single, large area (nearly 4 mm2) photodetector.
The integrated circuit biochips of the present invention also further comprise an integrated circuit that includes an optical transducer and associated optics and circuitry for generating an electrical signal in response to light or other radiation indicative of the presence of a target biological species, particularly a nucleic acid. The chip may also include a support for inmmobilizing a bioprobe, which is preferably a nucleic acid. In particular embodiments, a target nucleic acid may be tagged or labeled with a substance that emits a detectable signal; for example, luminescence. Alternatively, the bioprobe attached to the immobilized bioprobe may be tagged or labeled with a substance that emits a detectable or altered signal when combined with the target nucleic acid. The tagged or labeled species may be fluorescent, phosphorescent, or otherwise luminescent, or it may emit Raman energy or it may absorb energy.
The highly integrated biosensors of the present invention are advantageous in part because of fabricating multiple optical sensing elements and microelectronics on a single integrated circuit, and further combining the chip in preferred embodiments with a plurality of molecular hybridization probes (Geiger et al., 1990; Aubert et al., 1998). When the probes selectively bind to a targeted species, a signal is generated that is picked up by the chip. The signal may then be processed in several ways, depending on the nature of the signal.
In one aspect, the present invention concerns an integrated system that includes (1) a targeted nucleic acid sequence in combination with a biological probe which is modified to receive light or other radiation of a first frequency and thereby to emit light or other radiation of a different frequency than the first frequency, and (2) to detect the emitted radiation by means of a phototransducer. The target nucleic acid is typically a uniquely characteristic gene sequence of a pathogen such as a fungus, bacteria, or virus, or other distinct nucleic acid species such as may be found in mutant mammalian cells or in individuals with inherited errors of metabolism. The target nucleic acid is modified or labeled to include a tag or label that emits a signal upon exposure to an incident light or other radiation.
The target nucleic acid may be immobilized onto the integrated microchip that also supports a phototransducer and related detection circuitry. Alternatively, a gene probe may be immobilized onto a membrane or filter that is then attached to the microchip or to the detector surface itself. This approach avoids the need to bind the bioreceptor directly to the transducer and thus is attractive for simplifying large-scale production.
In one preferred embodiment of the invention, light of a highly directional or focused nature is impinged on a target nucleic that inherently or by virtue of an appropriate tag or label will emit a detectable signal upon irradiation. The irradiation may be provided by a suitable light source, such as a laser beam or a light-emitting diode (LED). With Raman, fluorescence or phosphorescence detection modes, the incident light is further kept separate from the emitted light using different light paths and/or appropriate optical filters to block the incident light from the detector.
For example, in the detection of a polynucleotide, one or more target nucleic acid sequence(s) are preferably hybridized with a nucleic acid sequence that is selected for that purpose (bioprobe). The selected bioprobe(s) are immobilized on a suitable substrate, either on the biochip itself or on a membrane type material that is then contacted or attached to the chip surface. The bioprobe may be labeled with a tag that is capable of emitting light or other non-radioactive energy. Upon hybridization with a target nucleic acid sequence, the hybrid product can be irradiated with light of suitable wavelength to emit a signal in proportion to the amount of target nucleic acid hybridized. The labeled bioprobe may comprise a labeled molecular bioreceptor. Known receptors are advantageous to use because of their known ability to selectively bind with the target nucleic acid sequence. In certain particular examples, the bioreceptor itself may exhibit changes in light emission when its cognate is bound.
In certain applications, it may be desirable to increase the amount of biotarget when only trace quantities are present in a sample. For example, the disclosed biochips are compatible for use with polymerase chain reaction (PCR(trademark)), a well-known technique used in amplifying polynucleotide sequences.
There are several methods for selectively identifying biological species, including antibody detection and assay as in the well-known enzyme-linked immuno-suppressant assays (ELISAs) employing molecular hybridization techniques. Generally speaking, it is possible to identify sequence-specific nucleic acid segments, and to design sequences complementary to those segments, thereby creating a specific probe for a target cell, such as different pathogen cells or even mammalian cells that have mutated from their normal counterparts. In principle, one can design complementary sequences to any identified nucleic acid segment. In many instances, unique sequences specific to an organism may be used as probes for a particular organism or cell type. The quantitative phenotypic analysis of yeast deletion mutants, for example, has utilized unique nucleic acid sequence identifiers to analyze deletion strains by hybridization with tagged probes using a high-density parallel array (Shoemaker et al., 1996).
Hybridization involves joining a single strand of nucleic acid with a complementary probe sequence. Hybridization of a nucleic acid probe to nucleic acid sequences such as gene sequences from bacteria, or viral DNA offers a very high degree of accuracy for identifying nucleic acid sequences complementary to that of the probe. Nucleic acid strands tend to be paired to their complements in double-stranded structures. Thus, a single-stranded DNA molecule will seek out its complement in a complex mixture of DNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (e.g., gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridization or reassociation of two complementary DNA strands include temperature, contact time, salt concentration, the degree of mismatch between the base pairs, and the length and concentration of the target and probe sequences. In perhaps the simplest procedure, hybridization is performed on an immobilized target or a probe molecule attached on a solid surface such as a nitrocellulose or nylon membrane or a glass plate.